The invention relates to simple assay systems for monitoring and measuring the activity of chromatin remodeling enzymes and modulators of these enzymes.
Chromatin remodeling enzymes and enzyme complexes are composed of at least one polypeptide and interact with nucleosomes to either stabilize or destabilize the nucleosome structure. The action of a chromatin remodeling enzyme leads to the enhanced or decreased accessibility of nucleosomal DNA sequences and thus affects nuclear processes that utilize DNA as a substrate, e.g., transcription, replication, DNA repair, and DNA organization within the nucleus, as well as the regulators of these processes.
Examples of chromatin remodeling enzymes and enzyme complexes include the yeast SWI/SNF complex and its homologues, which have been found in mice, humans, and flies and are likely present in all or most eukaryotes. To date, besides the SWI/SNF complex, four other complexes have been described that display the capacity to remodel nucleosomes in an ATP-dependent fashion. These complexes are: the NUcleosome Remodeling Factor complex ("NURF," Tsukiyama, T. and Wu, C., Cell, 83:1011-1020, 1995); the CHRomatin Accessibility Complex ("CHRAC," Varga-Weisz et al., EMBO J., 14:2209-2216, 1995); the ATP utilizing Chromatin assembly and remodeling Factor complex ("ACF," Ito et al., Cell, 90:145-155, 1997); and the Remodels the Structure of Chromatin complex ("RSC," Cairns et al., Cell, 87:1249-1260, 1996). The former three have been found in Drosophila melanogaster and the latter in Saccharomyces cerevisiae. Besides these five known macromolecular nucleosome remodeling complexes, there are numerous other nuclear enzymes (and enzyme complexes) that have the capacity to covalently modify the histone components of the nucleosomes.
It also has been suggested that covalent modification of histones (e.g., acetylation, phosphorylation, methylation, and ubiquitination) can by itself result in chromatin remodeling, and that such histone modifications can affect the activity of the above ATP-dependent molecular nucleosome remodeling complexes. The study of these remodeling enzymes has become increasingly important as a way to analyze chromatin structure and its site-specific remodeling in the regulation of gene expression. Thus, the characterization of these enzymes or enzyme complexes, which appear to interact directly with nucleosomes to stabilize or destabilize nucleosome structure and thereby repress or activate gene expression, and their modulators, is necessary to understand basic transcriptional regulatory mechanisms such as the regulation of gene expression during developmental processes.
Chromatin, the structure into which DNA in eukaryotes is neatly packaged, contains DNA, RNA, and protein in a compact form in which the majority of DNA sequences are structurally inaccessible and functionally inactive. Within this mass of sequences are a minority of active sequences, access to which is also structurally inhibited. Chromatin, in its simplest state, the nucleosome core particle ("nucleosome"), consists of about 146 base pairs of DNA wrapped around a histone core, i.e., an octamer of two of each of four different histone proteins (H2A, H2B, H3, and H4). This nucleoprotein structure presents a natural barrier to the process of RNA transcription, inhibiting both the accessibility of the general transcription machinery to promoter sequences and the binding of upstream regulatory proteins. The nucleoprotein structure also presents a barrier to DNA binding proteins involved in other types of nuclear processes. As their name suggests, chromatin remodeling enzymes function to facilitate transcription by remodeling chromatin, i.e., by providing access to DNA that would otherwise remain inaccessible.
The prototype and best characterized of the nucleosome remodeling enzyme complexes is the yeast (S. cerevisiae) SWI/SNF complex, which consists of 11 tightly associated subunits. Treich et al., Mol. Cell. Biol., 15:4240-4248 (1995). Genetic studies in S. cerevisiae indicate that the SWI/SNF complex is required for the induction of a class of genes regulated at the transcriptional level and that it functions by antagonizing chromatin-mediated transcriptional repression. Burns, L. G. and Peterson, C. L., Biochim. Biophys. Acta, 1350:159-68 (1997).
Previous in vitro studies have established that the yeast SWI/SNF complex destabilizes nucleosomes in vitro and that ATP hydrolysis is required for this reaction to occur. Cote et al., Science, 265:53-60 (1994). Furthermore, while the SWI/SNF remodeling reaction involves a disruption of DNA-histone interactions, it does not by itself result in nucleosome displacement. Cote et al., Science, 265:53-60 (1994); Owen-Hughes et al., Science, 273:513-516 (1996). SWI/SNF activity can also lead to an enhanced affinity of transcription factors for their binding sites when these sites are incorporated into nucleosomes. Cote et al., Science, 265:53-60 (1994); Owen-Hughes et al., Science, 273:513-6 (1996); Utley et al., J. Biol. Chem., 272:12642-12649 (1997). Previous studies have also shown that the yeast SWI/SNF complex displays nanomolar affinity for DNA fragments longer than 200 base pairs and for synthetic four-way DNA junctions, moreover, ATPase activity is stimulated by these DNAs. Quinn et al., Nature, 379:844-847 (1996).